Japanese encephalitis virus (JEV) vaccine loaded biodegradable poly(L-lactide-co-glycolide) (PLGA) microspheres (MSs) were prepared by W/O/W solvent evaporation method to study the possibility for oral vaccination. The influence of several preparation parameters as stirring rate, types and concentration of emulsifier, PLGA concentration, etc. has been observed on size, size distribution and biodegradation. The mean MSs size decreased when the agitation speed and the concentration of emulsifier were increased, and when the PLGA concentration was decreased. The surface morphology of porous and nonporous JEV vaccine loaded PLGA MSs was prepared from polyvinylalcohol and sodium dodecyl sulfate as used emulsifiers, respectively. From the assay of lactic acid and scanning electron microscope observation, it can be suggested that the rate of biodegradation of nonporous MSs was slower than that of porous surface morphology due to lower the surface area. Mechanisms of the formation of porous and nonporous surface by different types of emulsifier, and the biodegradation of MSs have been proposed. Also, the size and size distribution of JEV vaccine loaded PLGA MSs were discussed to apply oral vaccination through the Peyer's patches across the gastrointestinal tract.